Isao Matsuoka

Mechanism and Role of High Density Lipoprotein-induced Activation of AMP-activated Protein Kinase in Endothelial Cells

The upstream signaling pathway leading to the activation of AMP-activated protein kinase (AMPK) by high density lipoprotein (HDL) and the role of AMPK in HDL-induced antiatherogenic actions were investigated. Experiments using genetic and pharmacological tools showed that HDL-induced activation of AMPK is dependent on both sphingosine 1-phosphate receptors and scavenger receptor class B type I through calcium/calmodulin-dependent protein kinase kinase and, for scavenger receptor class B type I system, additionally serine-threonine kinase LKB1 in human umbilical vein endothelial cells. HDL-induced activation of Akt and endothelial NO synthase, stimulation of migration, and inhibition of monocyte adhesion and adhesion molecule expression were dependent on AMPK activation. The inhibitory role of AMPK in the adhesion molecule expression and monocyte adhesion on endothelium of mouse aorta was confirmed in vivo and ex vivo. On the other hand, stimulation of ERK and proliferation were hardly affected by AMPK knockdown but completely inhibited by an N17Ras, whereas the dominant-negative Ras was ineffective for AMPK activation. In conclusion, dual HDL receptor systems differentially regulate AMPK activity through calcium/calmodulin-dependent protein kinase kinase and/or LKB1. Several HDL-induced antiatherogenic actions are regulated by AMPK, but proliferation-related actions are regulated by Ras rather than AMPK.

Transcripts expressed using a bicistronic vector pIREShyg2 are sensitized to nonsense-mediated mRNA decay

BackgroundpIREShyg2 has been widely used as a bicistronic expression vector. However, it is not known if the vector would affect the expression of cloned genes via nonsense-mediated mRNA decay (NMD), an mRNA surveillance system that degrades mRNA with a premature termination codon (PTC). In mammalian cells, the induction of NMD requires either a long 3UTR or the presence of an exon-junction complex downstream of a PTC. The efficiency of NMD is greater when a PTC generates longer 3UTR. pIREShyg2 provides the first cistron gene with a long 3UTR consisting of a downstream intervening sequence (IVS), an internal ribosomal entry site (IRES) and the second cistron. Therefore, we hypothesized that the first cistron genes in pIREShyg2 are sensitized to NMD, which affects their expression levels. To examine this hypothesis, cDNAs encoding human granulocyte-macrophage colony-stimulating factor receptor β chain (βc) and its splice variant (βc79), in which the retention of a 79-base intron caused a frameshift generating 18 PTCs, were cloned into pIREShyg2 and stably expressed in a murine cell line, Ba/F3.ResultsCompared with wild-type βc, the mRNA levels of βc79 were less than one tenth and decayed faster. Both translation inhibition and Upf1 knockdown led to significantly greater up-regulation of βc79 than wild-type βc. However, the use of a monocistronic pMT21 vector abolished the up-regulatory effects of translation inhibition and Upf1 knockdown on both wild-type βc and βc79, suggesting that the NMD is attributable to a structural determinant in pIREShyg2. The elimination of the intron and the proximal 3 17 PTCs did not alter the greater effects of translation inhibition on βc79, suggesting that the first PTC, which determines 3UTR length, was sufficient to enhance NMD efficiency. Thus, transcripts of PTC-harboring genes with longer 3UTR are more efficiently degraded by the vector-dependent NMD than those of wild-type genes with relatively shorter 3UTR, resulting in minimized expression of truncated mutants.ConclusionsWe conclude that pIREShyg2, which sensitizes its bicistronic transcripts to NMD, may be useful for studying NMD but should be avoided when maximum expressions of PTC-harboring genes are required.

Divergent regulatory roles of extracellular ATP in the degranulation response of mouse bone marrow-derived mast cells

&NA; Mast cells (MCs) play a critical role in allergic inflammation. Although purinergic signalling is implicated in the regulation of various immune responses, its role in MC function is not fully understood. In this study, we investigated the regulatory role of purinergic signalling in MC degranulation, using mouse bone marrow‐derived mast cells (BMMCs). Notably, BMMCs expressed various functional P2 adenosine triphosphate (ATP) receptors, including ionotropic P2X4 and P2X7, involved in the regulation of BMMC degranulation. Thus, P2X7 receptor activation induced a marked degranulation from BMMCs directly. Although P2X4 receptor activation did not independently induce degranulation, it significantly potentiated the degranulation triggered by antigen‐induced, high‐affinity IgE receptor (Fc&egr;RI) stimulation. In addition, ATP synergistically augmented degranulation induced by adenosine A3 receptor activation. Moreover, BMMCs highly expressed ecto‐nucleotidase CD39, but not ecto‐5′‐nucleotidase (CD73), and were therefore unable to directly convert ATP to adenosine. However, in the presence of CD73‐expressing cells, ATP‐mediated BMMC stimulation caused a marked degranulation in a CD73‐ and adenosine‐dependent manner. These results demonstrate that purinergic signalling plays an important role in MC degranulation through at least three distinct mechanisms: (1) higher ATP concentrations directly induce degranulation via P2X7 receptor activation, (2) lower ATP concentrations augment Fc&egr;RI‐mediated degranulation via P2X4 receptor activation, and (3) in an ecto‐nucleotidase‐enrich environment, ATP and the converted product adenosine induce a synergistic degranulation by P1 and P2 receptor co‐activation. HighlightsRoles of purinergic signalling in mast cell degranulation were examined.P2X7 receptor activation directly induced mast cell degranulation.ATP potentiated antigen‐induced degranulation via P2X4 receptor activation.ATP and its degradation product adenosine synergistically induced degranulation.These purinergic regulations may be crucial for pathophysiological role of mast cells.

P2X7 receptor antagonist activity of the anti-allergic agent oxatomide

Activation of the P2X7 receptor by extracellular ATP is associated with various immune responses including allergic inflammation. Anti-allergic agents, such as H1-antihistamines, are known to inhibit the effects of different chemical mediators such as acetylcholine and platelet-activating factor. Therefore, we hypothesized that some anti-allergic agents might affect P2X7 receptor function. Using N18TG2 and J774 cells, which express functional P2X7 receptors, the effects of several anti-allergic agents on P2X7 receptor function were investigated by monitoring the ATP-induced increase in intracellular Ca(2+) concentrations ([Ca(2+)]i). Among the various agents tested, oxatomide significantly inhibited P2X7 receptor-mediated [Ca(2+)]i elevation in a concentration-dependent manner without affecting the P2Y2 receptor-mediated response in both N18TG2 and J774 cells. Consistently, oxatomide inhibited P2X7 receptor-mediated membrane current and downstream responses such as mitogen-activated protein kinase activation, inflammation-related gene induction, and cell death. In addition, oxatomide inhibited P2X7 receptor-mediated degranulation in mouse bone marrow-derived mast cells. Whole cell patch clamp analyses in HEK293 cells expressing human, mouse, and rat P2X7 receptors revealed that the inhibitory effect of oxatomide on ATP-induced current was most prominent for the human P2X7 receptor and almost non-existent for the rat P2X7 receptor. The potent inhibitory effects of oxatomide on human P2X7 receptor-mediated function were confirmed in RPMI8226 human B cell-like myeloma cells, which endogenously express the P2X7 receptor. Our results demonstrated that the antihistamine oxatomide also acts as a P2X7 receptor antagonist. Future studies should thus evaluate whether P2X7 receptor antagonism contributes to the anti-allergic effects of oxatomide.

Identification of novel selective P2Y6 receptor antagonists by high-throughput screening assay

Aims: The P2Y6 nucleotide receptor is widely involved in inflammatory responses, and is a promising molecular target for the treatment of inflammatory diseases. Although several P2Y6 receptor antagonists have been developed and evaluated thus far, none has successfully been developed into a therapeutic drug. In this study, we explored new promising compounds that inhibit the human P2Y6 receptor. Main methods: High‐throughput screening (HTS) was used to study the effects of various compounds on human P2Y6 receptors expressed in 1321N1 human astrocytoma cells by monitoring intracellular Ca2 + concentration ([Ca2 +]i) levels using an FDSS7000 real‐time fluorescence detector. IL‐8 concentration was measured by enzyme‐linked immunosorbent assay. Key findings: Among structurally diverse chemical libraries totalling 141,700 compounds, 43 compounds with an inhibitory activity against the P2Y6 receptor were identified. Further studies using a dose‐response assay, receptor selectivity assay, and chemokine measurement assay revealed the selective P2Y6 receptor inhibitor TIM‐38, which inhibited UDP‐induced [Ca2 +]i elevation in a dose‐dependent manner. TIM‐38 had an IC50 value of 4.3 &mgr;M and inhibited P2Y6 without affecting the response induced by four other human P2Y or muscarinic receptors. In addition, TIM‐38 inhibited UDP‐induced interleukin‐8 release in a dose‐dependent manner without affecting releases caused by other stimulus such as interleukin‐1&bgr; or tumour necrosis factor‐&agr;. Analyses of TIM‐38 derivatives revealed that the nitro moiety is vital to P2Y6 receptor inhibition. Significance: TIM‐38 acts as a novel structural antagonist of P2Y6 receptor and may be a good lead compound for developing a P2Y6 receptor‐targeted anti‐inflammatory drug.

Inhibition of P2Y6 receptor-mediated phospholipase C activation and Ca2+ signalling by prostaglandin E2 in J774 murine macrophages

Extracellular nucleotides act as inflammatory mediators through activation of multiple purinoceptors. Under inflammatory conditions, the purinergic signalling is affected by various inflammatory mediators. We previously showed that prostaglandin (PG) E2 suppressed the elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) stimulated by P2X4, P2Y2, and P2Y6 receptors in J774 murine macrophages. In this study, we examined the mechanism of PGE2 inhibitory effects on P2Y6 receptor-mediated function in J774 cells. The P2Y6 receptor agonist UDP induced a sustained elevation of [Ca(2+)]i by stimulating the phospholipase C (PLC) signalling pathway. PGE2 inhibited [Ca(2+)]i elevation and phosphatidylinositol (PI) hydrolysis in a concentration-dependent manner. J774 cells highly expressed the E-type prostanoid 2 (EP2) receptor subtype, a Gs-coupled receptor. PGE2 and a selective EP2 receptor agonist caused cyclic AMP (cAMP) accumulation in J774 cells. The inhibitory effects of PGE2 on P2Y6 receptor-mediated responses were mimicked by the selective EP2 receptor agonist. Although EP2 receptor is linked to adenylyl cyclase activation, PGE2-induced inhibition of Ca(2+) response and PI hydrolysis could not be mimicked by a lipophilic cAMP derivative, dibutyryl cAMP, or an adenylyl cyclase activator, forskolin. The inhibition of UDP-induced PLC activation by PGE2 was not affected by down-regulation of protein kinase C by phorbol-12-myristate-13-acetate treatment. PGE2 inhibited PLC activation induced by aluminium fluoride, but not by the Ca(2+)-ionophore, ionomycin. Finally, the inhibition of UDP-induced PLC activation by PGE2 was impaired by Gs knockdown using siRNA. These results suggest that EP2 receptor activation in macrophages negatively controls the Gq/11-PLC signalling through a Gs-mediated, but cAMP-independent signalling mechanism.

Effects of dexamethasone on purinergic signaling in murine mast cells: Selective suppression of P2X7 receptor expression

Mast cells express many different purinergic receptors, including ionotropic P2X4 and P2X7, which recognize the accumulation of extracellular ATP released from activated and/or damaged cells. This results in the stimulation of mast cell functions. In this study, we investigated the effects of dexamethasone (Dex), an anti-inflammatory glucocorticoid widely used for the treatment of allergic disease, on purinergic receptor expression in mouse bone marrow-derived mast cells (BMMCs). Treatment of BMMCs with Dex decreased P2X7 receptor mRNA levels in a time- and concentration-dependent manner without affecting the expression of other purinergic receptor subtypes. Accordingly, fluorescence-activated cell sorting analysis revealed that Dex treatment also decreased P2X7 receptor protein levels. This effect was mimicked by prednisolone, another anti-inflammatory glucocorticoid, and was inhibited by the glucocorticoid receptor antagonist mifepristone. Functionally, treatment of BMMCs with Dex impaired the P2X7-mediated rise in intracellular Ca2+ concentration, degranulation, and ethidium uptake, a response relevant to receptor-pore formation. Finally, oral administration of Dex to C57BL/6 mice inxa0vivo resulted in a significant decrease in P2X7 receptor expression in peritoneal mast cells. These results suggest that reduction of P2X7 receptor expression in mast cells might be one of the anti-allergic mechanisms of Dex.

Functional coupling between adenosine A 1 receptors and G-proteins in rat and postmortem human brain membranes determined with conventional guanosine-5′- O -(3-[ 35 S]thio)triphosphate ([ 35 S]GTPγS) binding or [ 35 S]GTPγS/immunoprecipitation assay

Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A1 receptor and G-protein was assessed by means of two [35S]GTPγS binding assays, i.e., conventional filtration method and [35S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A1 receptor-mediated Gαi-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [35S]GTPγS binding determined with conventional assay derives from functional activation of Gαi/o proteins (not restricted only to Gαi-3) coupled to adenosine A1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [35S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %Emax values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A1 receptor/G-protein interaction in postmortem human brain tissue.

Modulation pathways of NCX mRNA stability: involvement of RhoB.

Abstract:u2002 Cardiac Na+/Ca2+ exchanger 1 (NCX1) expression levels change under various pathophysiological conditions. However, its mechanism is unknown. We found that fluvastatin, an HMG‐CoA reductase inhibitor, decreased NCX1 mRNA and protein by inhibiting a small G protein, RhoB, in H9c2 cardiomyoblasts. Conversely, lysophosphatidylcholine (LPC) increased NCX1 mRNA and protein by activating RhoB. The effect of LPC was mediated by geranylgeranylation but not farnesylation of RhoB. Furthermore, we also detected that activation of RhoB increased NCX1 mRNA stability. Our results suggest that RhoB is involved in modulation of cardiac NCX1 mRNA expression.